CN113528536B - Wheat spike length increasing gene HL1 and application thereof - Google Patents

Abstract

The invention discloses a wheat spike length increasing gene HL1 and application thereof. An application of wheat spike length increasing gene HL1 in improving wheat spike length by genetic engineering means. The wheat spike length increasing gene HL1 provided by the invention is transferred into plants by a genetic engineering method, so that the spike length of the plants can be increased. Because the gene is an endogenous gene existing in the wheat of the grain crops, the transfer of the gene does not influence the food safety of plants, and can be widely applied to the breeding process of increasing the spike length of various plants.

Description

Wheat spike length increasing gene HL1 and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to a wheat spike length increasing gene HL1 and application thereof.

Background

Wheat is one of main grain crops in China, the yield of the wheat is related to national grain safety, and the improvement of the yield is always the most main goal of wheat breeders. The ear is the place where wheat seeds grow, is closely related to the grain weight and the ear grain number in the yield components, directly influences the yield, and is paid attention and paid attention to by breeders in the variety breeding process. The ideal spike shape has important function for improving the unit yield of wheat. The tassel density is an important trait of the tassel and is an important factor in determining the tassel type (Donmez et al 2001; kumar et al 2007). In addition, a smaller spike density may reduce the severity of scab (Suzuki et al 2012; jones et al 2018; sun Yangyang et al, 2017). Therefore, understanding the genetic basis of the spike density and the molecular mechanism formed are of great significance to the genetic improvement of wheat and the promotion of yield improvement.

The spike density is a quantitative trait controlled by multiple genes, the genetic mechanism is complex and is mainly controlled by an additive effector gene, and the generalized genetic rate is as high as 69.7-97.9% (Heidari et al 2011; xu et al 2014; zhai et al 2016). At present, the research on wheat spike density is focused on a QTL positioning stage, and QTL for controlling spike density is detected on 21 chromosomes (Sourdille et al 2000,2003; marza et al 2006; ma et al 2007; heidari et al 2011; mazen et al 2014; xu et al 2014; zhai et al 2016; liu et al 2018). QTLs controlling the spike density have both a minor QTL and a major QTL, which explain phenotypic variation varying from 2.0% to 29.9%.

Qcpt.nau-2D is a major QTL for controlling spike density detected on the 2DS chromosome by the present laboratory early stage using Nada 2419 XWang water white recombinant inbred line population and is located between Xcfd53-DG371 of 0.9cM (Wu et al 2013). This site was detected in several germplasm as being correlated with tassel density, accounting for up to 29.9% of phenotypic variation (Sourdille et al 2003; heidari et al 2011; zhai et al 2016; chai et al 2018).

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and providing a gene HL1 for controlling the spike length of wheat.

It is a further object of the invention to provide the use of the HL1 gene.

The aim of the invention can be achieved by the following technical scheme:

the HL1 gene is determined to be a gene for controlling the spike length, and the nucleotide sequence is shown as SEQ ID NO. 1.

Recombinant expression vector containing wheat spike length controlling gene HL1.

The recombinant expression vector is preferably a vector of which the starting vector is the HL1 gene driven by a self promoter.

The wheat control spike length gene HL1 is applied to the improvement of wheat varieties; preferably the gene is used to increase wheat ear length.

The recombinant expression vector is applied to the improvement of wheat varieties; preferably the recombinant expression vector is used for increasing the wheat ear length.

The beneficial effects are that:

we further mapped this QTL to the Xwgrc1257-Xwgrc1366 interval, which was physically separated from Xmag9642 and Xwgrc1808 by a secondary segregating population, and was approximately 100kb in physical distance. Through sequence comparison among parents, the candidate gene of HL1 is determined to be Qcpt. The invention provides a novel gene HL1 for increasing spike length of wheat. The spike length increasing gene HL1 provided by the invention is transferred into wheat through transgenosis, so that the spike length of the wheat can be increased. Because the gene is an endogenous gene existing in the wheat of the grain crops, the existence of the gene does not influence the food safety of plants, and the gene can be applied to crop breeding.

Drawings

FIG. 1 PCR detection of transgenic wheat.

Wherein A is a positive control plasmid carrying the HL1 gene; b is Mianyang 99-323 of trans-HL 1 gene; c is transgenic experimental receptor material Mianyang 99-323, figure 2 ear of transgenic wheat

Wherein the left side is transgenic experimental receptor material Miyang 99-323, and the right side is transgenic HL1 gene Miyang 99-323

Detailed Description

The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the examples are described only for illustrating the invention and that the invention described in detail in the claims should not be limited either.

Example 1: determination of HL1 gene.

Hybridization of Mianyang 99-323 with Mianyang 99-323 near isogenic line carrying Qcpt.nau-2D (Wu et al 2014) constructed a secondary F2 population, screening with Cfd53 (SEQ ID NO.2/SEQ ID NO. 3) and DG371 (SEQ ID NO.4/SEQ ID NO. 5) markers, obtaining individuals that recombine between these two markers. Qcpt.nau-2D was defined in the 100kb interval between Xwgrc1257 (SEQ ID NO.6/SEQ ID NO. 7) and Xwgrc1366 (SEQ ID NO.8/SEQ ID NO. 9) depending on the genotype and spike length phenotype of the recombinants. Only one coding gene with complete transcripts exists in the region carrying Qcpt.nau-2D Mianyang 99-323 near isogenic line, which is 2427bp long, namely HL1. The gene length is 2426bp corresponding to the same position of Mianyang 99-323. Compared to HL1 in the near isogenic line, the Mianyang 99-323 gene sequence has one SNP and one INDEL variation, both of which are located adjacently, which results in premature termination of translation in Mianyang 99-323. From this, it was inferred that HL1 was a candidate gene for Qcpt.nau-2D.

Example 2: obtaining cDNA sequence of encoding Ta FHB1 protein.

The total RNA of wheat ears is extracted by using Trizol kit of the Johnst company by taking small ear cDNA of Nandina 2419 in the tillering stage as a template, and reverse transcription is carried out by using reverse transcription kit of the Promega company to synthesize single-stranded cDNA. PCR was performed using primer P1 (SEQ ID NO.10/SEQ ID NO. 11) in a 25. Mu.L amplification system comprising 5ng template, 5pmol each of F and R primers, dATP, dTTP, dCTP and dGTP each of 5nmol,37.3nmol MgCl2,0.5 units of DNA polymerase, 1 XPCR buffer. The amplification procedure was denaturation at 94℃for 3 min; 30 cycles, denaturation at 94℃for 20 seconds, annealing at 60℃for 30 seconds, and extension at 72℃for 2 minutes; finally, the extension is carried out at 72 ℃ for 10 minutes. A2427 bp nucleotide sequence containing an open reading frame was obtained by PCR amplification, and the nucleotide was ligated with the T-vector at 16℃for 30min in a ligation system of 10. Mu.L, including 1. Mu.L of the T-vector, 4. Mu.L of the PCR product, 1. Mu.L of ligase buffer, and ddH2O 3. Mu.L. The ligation product was transformed into E.coli cells by heat shock at 42℃for 40 seconds and sent to Huada gene company for sequencing, and the obtained sequence was shown in SEQ ID NO. 1.

Example 3: spike length increase of wheat transformed with HL1 gene

PCR amplification is carried out by taking Nanguo 2419 genome DNA as a template and utilizing a primer P2 (SEQ ID NO.12/SEQ ID NO. 13), and the PCR product obtained by amplification is subjected to enzyme digestion by using restriction enzyme HindIII and then is connected with a pUCBS expression vector subjected to enzyme digestion by using the same restriction enzyme, so that an HL1 gene gun stable transformation expression vector is formed. The constructed vector was transformed into DH 5. Alpha. E.coli strain by heat shock method, and positive clones were obtained by selection in LB medium containing 50. Mu.g/mL kanamycin. Referring to the materials and methods published by Weeks in 1993 on page 1077-1084 of plant Physiol at 102 th stage, callus which is produced by short-term culture of young embryo and can regenerate with higher frequency into fertile plants is taken as a receptor, HL1 gene under the control of a promoter is introduced into Mianyang 99-323 of short-spike wheat by a gene gun method, the callus after gene gun transformation is placed on an MS culture medium containing 20mg/LG418 for screening, seedlings are once screened on an MS culture medium containing 30mg/L and 40mg/L after differentiation, finally obtained regenerated seedlings are transferred into an MS culture medium containing 1mg/LNAA, leaves are cut after roots grow out, DNA is extracted for PCR detection, and transgenic wheat carrying HL1 is obtained (figure 1). The ear length of transgenic wheat was significantly increased (fig. 2).

Sequence listing

<110> Nanjing agricultural university

<120> a wheat spike length increasing gene HL1 and application thereof

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caccacgtca tggtcgccga cccggccggg tactccccga cgccgcccac cccgcccggg 660

atgcccgcgc ccaggcagct gctctccggc ggccggggcg acctcgcccc gctcgccatg 720

ctggaggaca gcgtcaagcg cctcaagtcg ccctcggcgt cgccgggcgc gatgctgccg 780

cggccgcagg ccgaggccgc gctctcgctg ctcgccgact ggttcctcga gtcgtccggc 840

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atcgtcgccg accgcttcgg ctccaagact ctgcgtgatc tcgagaccaa gcaccattgg 1260

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gtgaaggaga tggagaccga gaggcccctg gtcgggcaat gcctgccgct ctgggaggac 1740

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gtggatccct cctcctcctc agtgtag 2427

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ccctatttcc cccatgtctt 20

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aaggagggca catatcgttg 20

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gtacaagctt aacggcgaaa caacccttac c 31

Claims (3)

1. Wheat spike length control geneHL1The application of the nucleotide sequence in increasing the wheat ear length is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. Comprising the gene as claimed in claim 1HL1The recombinant expression vector of (C) is applied to increasing the wheat spike length.

3. The use according to claim 2, wherein the starting vector of the recombinant expression vector is a geneHL1Vectors under the promoter of their own promoters.